Bioprocess Biosyst Eng (2017) 40:911–918 DOI 10.1007/s00449-017-1755-5
RESEARCH PAPER
Extraction of microalgae derived lipids with supercritical carbon dioxide in an industrial relevant pilot plant Jan Lorenzen1 · Nadine Igl2 · Marlene Tippelt2 · Andrea Stege3 · Farah Qoura1 · Ulrich Sohling3 · Thomas Brück1
Received: 2 December 2016 / Accepted: 21 February 2017 / Published online: 15 March 2017 © The Author(s) 2017. This article is published with open access at Springerlink.com
Abstract Microalgae are capable of producing up to 70% w/w triglycerides with respect to their dry cell weight. Since microalgae utilize the greenhouse gas C O2, they can be cultivated on marginal lands and grow up to ten times faster than terrestrial plants, the generation of algae oils is a promising option for the development of sustainable bioprocesses, that are of interest for the chemical lubricant, cosmetic and food industry. For the first time we have carried out the optimization of supercritical carbon dioxide (SCCO2) mediated lipid extraction from biomass of the microalgae Scenedesmus obliquus and Scenedesmus obtusiusculus under industrrially relevant conditions. All experiments were carried out in an industrial pilot plant setting, according to current ATEX directives, with batch sizes up to 1.3 kg. Different combinations of pressure (7–80 MPa), temperature (20–200 °C) and C O2 to biomass ratio (20– 200) have been tested on the dried biomass. The most efficient conditions were found to be 12 MPa pressure, a temperature of 20 °C and a C O2 to biomass ratio of 100, resulting in a high extraction efficiency of up to 92%. Since the optimized C O2 extraction still yields a crude triglyceride product that contains various algae derived contaminants, such as chlorophyll and carotenoids, a very effective and scalable purification procedure, based on cost efficient bentonite based adsorbers, was devised. In addition to the * Jan Lorenzen [email protected] 1
Department of Chemistry, Technical University of Munich, Lichtenbergstrasse 4, 85748 Garching, Germany
2
Hopfenveredlung St. Johann GmbH & Co. KG, Auenstr. 18‑20, 85283 Wolnzach, Germany
3
Clariant Produkte (Deutschland) GmbH, Ostenrieder Str. 15, 85368 Moosburg, Germany
sequential extraction and purification procedure, we present a consolidated online-bleaching procedure for algae derived oils that is realized within the supercritical C O2 extraction plant. Keywords Supercritical carbon dioxide extraction · Microalgae · Scenedesmus · Lipids · Bentonite Abbreviations SCCO2 Supercritical carbon dioxide PUFA Polyunsaturated fatty acid FA Fatty acid MPa Mega pascal FAME Fatty acid methyl ester
Introduction Recently, governmental CO2 emission regulations and an increased awareness of sustainability drive the development of renewable feedstocks based industrial processes. Hence, many different renewable feedstocks have been tested in the last decades, like organic waste, microbe-derived lipids or different types of plant seeds. With respect to sustainability and lipid productivity, microalgae are deemed to be one of the most relevant feedstocks for lipid type chemical products [1–3]. Conservative estimations postulate that about 72.000 algae species exist [4], most of them represented by microalgae. Compared to other renewable feedstocks for bio-lipid production, like rapeseed or soybeans, microalgae show several beneficial characteristics. In contrast to vascular plants, microalgae exhibit high growth rates at low area consumption. Lipid contents higher than 50% are reported for various species and can be controlled by the composition of the cultivation medium. A nitrogen starvation, for
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example, leads to a significant increase in lipid-storage in different microalgae [5–7]. In addition to these arguments, one of the most important advantage of microalgae is the lack of competition with agricultural activities. In this context, it is also important to mention that many lipid-producing microalgae can be cultivated in brackish or salt water, which secures valuable freshwater resources for human activity. One of the main obstacles to overcome in the usage of microalgae derived lipids is to find a method for lipid extraction that is efficient, economically relevant and environmentally friendly. One of the most common lipid extraction methodologies is unspecific organic solvent extraction according to the work of Bligh and Dyer [8]. Other methods combine solvent and/ or enzyme assisted techniques that are either highly toxic (n-hexane, methanol) or energetically inefficient [9]. By contrast, lipid extraction with supercritical fluids, especially supercritical carbon dioxide, has recently risen as a powerful industrial tool for environmentally friendly lipid recovery from biomass. Recently, many reports have discussed the extraction of microalgae lipids with supercritical carbon dioxide in small pilot plants, in the majority of the cases with a polar co-solvent (e.g., ethanol or water [10, 11]), an approach that cannot be transformed to industrial scale due to legislative safety regulations. In this study, we optimized the performance of an industrially relevant supercritical carbon dioxide extraction process for the recovery of lipids from dried microalgae biomass for the first time. The extraction of microalgae lipids with supercritical carbon dioxide was performed in a pilot plant according to European industrial standards for large
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Bioprocess Biosyst Eng (2017) 40:911–918
scale supercritical fluid extractions (ATEX directives). Furthermore, we present a subsequent purification procedure for the crude microalgae lipid extracts, to provide a lipid fraction that is applicable for bio-lubricant/bio-fuel and cosmetic applications.
Materials and methods Strains and cultivation Two different batches of algae biomass have been analyzed. The first batch was provided by Hochschule Anhalt, a heterologous culture consisting of 85–90% Scenedesmus obliquus, low percentages of Chlorella vulgaris and Chlorella kessleri as well as traces of Chlorella vacuolatus. The algae were cultivated under non-limiting conditions with natural sunlight illumination. The second batch analyzed was an unialgal culture of Scenedesmus obtusiusculus, cultivated in BG-11 medium under non-limiting conditions, using LED-assisted natural sunlight illumination. After the cultivation period, the cells were harvested from the cultivation broth, cracked by high pressure homogenization and lyophilized. Conventional solvent extraction The conventional extraction of lipids from microalgae biomass was performed according to Bligh and Dyer [8], with hexane as single solvent for 8 h. Supercritical carbon dioxide (SCCO2) extraction
Bioprocess Biosyst Eng (2017) 40:911–918
Liquid CO2 from the storage tank (ST) is pressurized by a O2-pump and heated to the extraction temperature by a C heat exchanger (HEX 1). Subsequently, the CO2 flows in the extraction vessel (EX1) containing the biomass. The CO2/extract mixture is separated by controlled pressure reduction under simultaneous warming by heat exchangers (HEX2, HEX3) into a C O2 vapour and an extract phase (separators SP1, SP2). The extracts are removed from the process while the gaseous C O2 remains in the process. Afterwards, the CO2 is liquefied by a condenser and recirculated. All extractions were performed with sample sizes from 650 to 1300 g of lyophilized algae biomass. In terms of the high amount of biomass required for the extraction in the pilot plant, not all experiments could be carried out in duplicates. The online bentonite bleaching was only tested on the biomass of the unialgal S. obtusiusculus culture. The tests were focused on this strain, because it showed the most promising process parameters in terms of cultivation stability and biomass yields and therefore became the leading strain in the Advanced Biomass Value (ABV) project.
913 Table 1 Adapted temperature and pressure profiles of astaxanthin extractions, applied to mixed, lyophilized Scenedesmus biomass and the corresponding extraction yields after S CCO2 extraction and soxhlet extraction of the spent material Extraction pressure (MPa)
Extraction CO2: temp. (°C) biomass ratio
Extraction yield (% w/w)
Soxhlet yields of spent material (% w/w)
30 50 60 80
50 60 60 80
6.9 6.7 5.8 7.6
1.2 0.5 0.9 0.2
100 100 200 100
Extraction time 540 min
110 °C for 1 h, the oil was analyzed via inductively coupled plasma optical emission spectroscopy (ICP-OES) with a Perkin Elmer Optima 3300 DV, analog to DIN EN ISO 11885. The samples were dissolved in kerosene.
Results
Purification and analysis of microalgae extracts
Effect of pressure and temperature on extraction yield
Lipid analysis
The major points of regulation during the process of SCCO2 extraction are changes in extraction temperature and the applied pressure. For the first experiments, temperature and pressure profiles established in the pilot plant from astaxanthin extraction were tested on the lyophilized biomass of the mixed Scenedesmus culture (Table 1). The different temperature and pressure profiles resulted in comparable quantities of extracted lipids, but showed large differences in the quality of the extracts. The extracts obtained from the profiles with lower temperature and pressure were unclear, greenish-brownish, viscous liquids, whereas the extracts gained from the profiles with high temperature and pressure appeared as very dark brownish, almost black and extremely viscous substance. For most of the downstream applications of the extracted algae lipid fractions, a clear and homogeneous extract is required. In terms of developing an economically and ecologically balanced extraction process and to obtain a more clear and homogeneous extract, the following experiments were carried out under milder conditions (Table 2). The milder extraction conditions applied to the biomass samples resulted in lipid yields between 6.5 and 8.3% (w/w), suggesting that there is no statistically relevant distinction in the extraction efficiency compared to the extraction profiles applied before (Table 1). Although equal quantities were obtained, the quality of the extracts strongly improved. The obtained extracts are clear, homogenous liquids with slightly different colors. The extracts from the profiles 1 and 3 had a greenish color, while the extracts
The direct transesterification of the algae derived lipids was performed according to a modified protocol of Griffiths et al. [12] with the following modifications: replacement of the C17-TAG by a C12-TAG, replacement of BF3 methanol by a HCL-methanol solution, and the C19-ME was omitted. Subsequently, the resulting fatty acid methyl ester (FAME) extract was injected into a Thermo Scientific™ TRACE™ Ultra Gas Chromatograph coupled to a Thermo DSQ™ II mass spectrometer and the Triplus™ Autosampler injector. Column: Stabilwax® fused silica capillary (30 m × 0,25 mm, film thickness 0.25 μm). (Program: initial column temperature 50 °C, increasing (4 °C/min) up to a final temperature of 250 °C. Carrier gas: hydrogen, flow rate 3.5 mL/min.) Peaks were identified by comparison to a marine oil standard (Restek) or by specific molecular masses detected. Purification of crude micro algae extracts The lipids were purified with an adsorbent based on montmorillonite. In a column with n-hexane 20 g Tonsil 5 10® from Clariant Produkte (Deutschland) GmbH were filled and given 10 min to swell in the solvent. 2–3 g algae oil were applied to the column and given time to sink completely into the adsorbent. The elution was done with n-hexane until 150 ml eluate was collected. After removing the solvent with a rotary evaporator and drying the oil at
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Table 2 Different extraction profiles applied to mixed, lyophilized Scenedesmus biomass and the corresponding extraction yields after S CCO2 extraction and soxhlet extraction of the spent material
Profile
Extraction pressure (MPa)
Extraction temp. (°C)
CO2: biomass ratio
Extraction yield (% w/w)
Soxhlet yields of spent material (% w/w)
1 2 3 4
7 7 12 12 12 15 15
20 20 20 20 20 20 20
20 100 20 100 100 100 100
6.5 6.6 6.6 8.3 7 6.6 6.5
1.0 2.0 1.8 2.1 2.8 3.0 2.8
5
Extraction time 540 min
Table 3 Extraction parameters applied to Scenedesmus obtusiusculus biomass (unialgal culture) and the corresponding extraction yields after SCCO2 extraction and soxhlet extraction of the spent material (in duplicate) Extraction pressure (MPa)
Extraction CO2: temp. (°C) biomass ratio
Extraction yield (% w/w)
Soxhlet yields of spent material (% w/w)
12 12
20 20
6.4 6.4
0.5 0.9
100 100
Extraction time 540 min
As a proof of concept the extraction profile was applied to an unialgal S. obtusiusculus culture (Table 3) and was used to monitor the extraction efficiency (Fig. 1). The transferability of the extraction profile to other microalgae species within the Scenedesmus family is depicted in Table 3. Both quantity and quality of the extracts were comparable to those of the mixed S. obliquus culture extracted previously. The extraction curves in Fig. 1 depict the extraction yields (in % w/w) of four different samples, two samples of a mixed S. obliquus culture (square markers) and two samples of an unialgal S. obtusiusculus culture (triangle markers), as a function of the C O2 to biomass ratio. The four extraction curves follow a sigmoidal function and are almost reaching a plateau of maximum lipid yield at a ratio of 100 (CO2/biomass), matching the results shown in Table 1. In addition, the technical duplicates of the extraction curves demonstrate that the extraction method is reproducible. Microalgae extract analysis Fatty acid composition of extracts obtained from different extraction profiles
Fig. 1 Algae lipid extraction yields from two samples of a mixed Scenedesmus obliquus culture (square markers) and from two samples of an unialgal Scenedesmus obtusiusculus culture (triangle markers), expressed as a function of increasing C O2 to biomass ratio. Extraction time 840 min
from the other three profiles showed a more brownish to orange color. The extraction profile 4 showed the best results in this experimental set-up, with respect to the extraction effency and sustainability, and was employed for subsequent extractions.
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The analysis of the fatty acid (FA) composition of the microalgae extracts (Table 4) showed no significant difference for the extraction profiles tested. The major compounds in these extracts are linolenic acid (C18:3), hexadecatetraenoic acid (C16:4), linoleic acid (C18:2), oleic acid (C18:1) and palmitic acid (C16:0). The FA profile of the unialgal culture of S. obtusiusculus was similar to the one from the mixed S. obliquus culture in its major components, but the percentage of FAs identified from the total lipid extract is reduced by 20–30%. The obtained FA profiles accord with previously published profiles.
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Table 4 Major fatty acid (FA) components in microalgae extracts from different extraction profiles, expressed as percentage of the total lipid extract (mean ± standard deviation) S. obliquus
S. obtusiusculus
Profile Pressure (MPa) CO2: biomass ratio C14 C16 C16:1 cis ω7 C16:1 ω9 C16:2 ω6 C16:3 ω3 C16:4 ω3 C18 C18:1 ω9 C18:1 ω7 C18:2 ω6 C18:3 ω3 C18:4 ω3
1 7 20 0.29 ± 0.01 6.30 ± 0.19 0.72 ± 0.02 1.40 ± 0.06 1.24 ± 0.04 2.31 ± 0.09 9.81 ± 0.33 0.13 ± 0.00 6.59 ± 0.14 1.20 ± 0.02 9.04 ± 0.18 24.78 ± 0.44 2.26 ± 0.09
2 7 100 0.27 ± 0.02 6.38 ± 0.09 0.72 ± 0.01 1.83 ± 0.02 1.31 ± 0.02 2.54 ± 0.02 11.93 ± 0.02 0.15 ± 0.00 7.02 ± 0.08 1.28 ± 0.01 9.75 ± 0.11 27.44 ± 0.24 2.81 ± 0.02
3 12 20 0.27 ± 0.01 6.46 ± 0.22 0.74 ± 0.03 1.53 ± 0.05 1.27 ± 0.04 2.28 ± 0.04 9.56 ± 0.55 0.14 ± 0.00 6.93 ± 0.23 1.25 ± 0.04 9.18 ± 0.05 24.75 ± 0.59 2.25 ± 0.12
4 12 100 0.29 ± 0.01 6.01 ± 0.05 0.74 ± 0.03 2.16 ± 0.03 1.43 ± 0.02 2.77 ± 0.03 13.25 ± 0.09 0.16 ± 0.00 7.43 ± 0.08 1.38 ± 0.04 10.20 ± 0.07 28.12 ± 0.16 3.02 ± 0.03
12 100 0.30 ± 0.01 5.68 ± 0.03 0.78 ± 0.02 2.03 ± 0.02 1.48 ± 0.07 2.77 ± 0.03 13.28 ± 0.44 0.15 ± 0.01 7.45 ± 0.07 1.38 ± 0.01 10.21 ± 0.08 28.44 ± 0.28 3.07 ± 0.01
5 15 100 0.29 ± 0.02 6.56 ± 0.09 0.77 ± 0.01 2.01 ± 0.00 1.44 ± 0.02 2.72 ± 0.02 12.66 ± 0.23 0.15 ± 0.00 7.24 ± 0.09 1.34 ± 0.02 9.94 ± 0.15 27.69 ± 0.35 2.96 ± 0.05
15 100 0.29 ± 0.02 6.07 ± 0.02 0.77 ± 0.01 1.89 ± 0.01 1.41 ± 0.01 2.70 ± 0.01 12.53 ± 0.16 0.14 ± 0.00 7.25 ± 0.07 1.34 ± 0.03 9.94 ± 0.10 27.75 ± 0.07 2.95 ± 0.01
C20 C20:1 ω9 FA yield
ND 0.05 ± 0.04 65.72 ± 1.58
ND 0.10 ± 0.01 73.53 ± 0.37
ND 0.05 ± 0.04 66.66 ± 0.83
0.03 ± 0.04 0.08 ± 0.00 77.07 ± 0.48
0.02 ± 0.00 0.08 ± 0.01 77.09 ± 0.87
0.02 ± 0.00 0.08 ± 0.01 75.87 ± 0.59
ND 0.10 ± 0.01 75.11 ± 0.09
4 12 100 0.16 ± 0.00 3.25 ± 0.05 0.45 ± 0.01 0.41 ± 0.03 0.71 ± 0.01 1.13 ± 0.02 3.91 ± 0.07 0.17 ± 0.01 4.09 ± 0.03 0.79 ± 0.00 6.93 ± 0.07 20.62 ± 0.29 0.88v ± 0.02 ND ND 43.52 ± 0.52
All extractions were carried out at 20 °C FA yield percentage of FAs from total lipids, ND not detectable
Table 5 Microalgae oils obtained from different extraction profiles before (crude) and after (purified) the processing with a bentonite
Processing of microalgae oils with bentonites The microalgae lipid fractions obtained from the mild extraction profiles show a higher quality then the ones obtained under harsh extraction conditions. Anyway the coloring of the extracts implements a relatively high amount of substances like chlorophylls and carotenoids. As
these substances could be problematic for any downstream usage of the microalgae oils, e.g., in technical applications, the lipid fractions were processed with the bentonite Tonsil 510®. The results of a single treatment of microalgae oils with the bentonite are shown in Table 5. After the processing the lipid fractions appear as a clear, liquid oil, suggesting that
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Cleaned
RESEARCH PAPER
Extraction of microalgae derived lipids with supercritical carbon dioxide in an industrial relevant pilot plant Jan Lorenzen1 · Nadine Igl2 · Marlene Tippelt2 · Andrea Stege3 · Farah Qoura1 · Ulrich Sohling3 · Thomas Brück1
Received: 2 December 2016 / Accepted: 21 February 2017 / Published online: 15 March 2017 © The Author(s) 2017. This article is published with open access at Springerlink.com
Abstract Microalgae are capable of producing up to 70% w/w triglycerides with respect to their dry cell weight. Since microalgae utilize the greenhouse gas C O2, they can be cultivated on marginal lands and grow up to ten times faster than terrestrial plants, the generation of algae oils is a promising option for the development of sustainable bioprocesses, that are of interest for the chemical lubricant, cosmetic and food industry. For the first time we have carried out the optimization of supercritical carbon dioxide (SCCO2) mediated lipid extraction from biomass of the microalgae Scenedesmus obliquus and Scenedesmus obtusiusculus under industrrially relevant conditions. All experiments were carried out in an industrial pilot plant setting, according to current ATEX directives, with batch sizes up to 1.3 kg. Different combinations of pressure (7–80 MPa), temperature (20–200 °C) and C O2 to biomass ratio (20– 200) have been tested on the dried biomass. The most efficient conditions were found to be 12 MPa pressure, a temperature of 20 °C and a C O2 to biomass ratio of 100, resulting in a high extraction efficiency of up to 92%. Since the optimized C O2 extraction still yields a crude triglyceride product that contains various algae derived contaminants, such as chlorophyll and carotenoids, a very effective and scalable purification procedure, based on cost efficient bentonite based adsorbers, was devised. In addition to the * Jan Lorenzen [email protected] 1
Department of Chemistry, Technical University of Munich, Lichtenbergstrasse 4, 85748 Garching, Germany
2
Hopfenveredlung St. Johann GmbH & Co. KG, Auenstr. 18‑20, 85283 Wolnzach, Germany
3
Clariant Produkte (Deutschland) GmbH, Ostenrieder Str. 15, 85368 Moosburg, Germany
sequential extraction and purification procedure, we present a consolidated online-bleaching procedure for algae derived oils that is realized within the supercritical C O2 extraction plant. Keywords Supercritical carbon dioxide extraction · Microalgae · Scenedesmus · Lipids · Bentonite Abbreviations SCCO2 Supercritical carbon dioxide PUFA Polyunsaturated fatty acid FA Fatty acid MPa Mega pascal FAME Fatty acid methyl ester
Introduction Recently, governmental CO2 emission regulations and an increased awareness of sustainability drive the development of renewable feedstocks based industrial processes. Hence, many different renewable feedstocks have been tested in the last decades, like organic waste, microbe-derived lipids or different types of plant seeds. With respect to sustainability and lipid productivity, microalgae are deemed to be one of the most relevant feedstocks for lipid type chemical products [1–3]. Conservative estimations postulate that about 72.000 algae species exist [4], most of them represented by microalgae. Compared to other renewable feedstocks for bio-lipid production, like rapeseed or soybeans, microalgae show several beneficial characteristics. In contrast to vascular plants, microalgae exhibit high growth rates at low area consumption. Lipid contents higher than 50% are reported for various species and can be controlled by the composition of the cultivation medium. A nitrogen starvation, for
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example, leads to a significant increase in lipid-storage in different microalgae [5–7]. In addition to these arguments, one of the most important advantage of microalgae is the lack of competition with agricultural activities. In this context, it is also important to mention that many lipid-producing microalgae can be cultivated in brackish or salt water, which secures valuable freshwater resources for human activity. One of the main obstacles to overcome in the usage of microalgae derived lipids is to find a method for lipid extraction that is efficient, economically relevant and environmentally friendly. One of the most common lipid extraction methodologies is unspecific organic solvent extraction according to the work of Bligh and Dyer [8]. Other methods combine solvent and/ or enzyme assisted techniques that are either highly toxic (n-hexane, methanol) or energetically inefficient [9]. By contrast, lipid extraction with supercritical fluids, especially supercritical carbon dioxide, has recently risen as a powerful industrial tool for environmentally friendly lipid recovery from biomass. Recently, many reports have discussed the extraction of microalgae lipids with supercritical carbon dioxide in small pilot plants, in the majority of the cases with a polar co-solvent (e.g., ethanol or water [10, 11]), an approach that cannot be transformed to industrial scale due to legislative safety regulations. In this study, we optimized the performance of an industrially relevant supercritical carbon dioxide extraction process for the recovery of lipids from dried microalgae biomass for the first time. The extraction of microalgae lipids with supercritical carbon dioxide was performed in a pilot plant according to European industrial standards for large
13
Bioprocess Biosyst Eng (2017) 40:911–918
scale supercritical fluid extractions (ATEX directives). Furthermore, we present a subsequent purification procedure for the crude microalgae lipid extracts, to provide a lipid fraction that is applicable for bio-lubricant/bio-fuel and cosmetic applications.
Materials and methods Strains and cultivation Two different batches of algae biomass have been analyzed. The first batch was provided by Hochschule Anhalt, a heterologous culture consisting of 85–90% Scenedesmus obliquus, low percentages of Chlorella vulgaris and Chlorella kessleri as well as traces of Chlorella vacuolatus. The algae were cultivated under non-limiting conditions with natural sunlight illumination. The second batch analyzed was an unialgal culture of Scenedesmus obtusiusculus, cultivated in BG-11 medium under non-limiting conditions, using LED-assisted natural sunlight illumination. After the cultivation period, the cells were harvested from the cultivation broth, cracked by high pressure homogenization and lyophilized. Conventional solvent extraction The conventional extraction of lipids from microalgae biomass was performed according to Bligh and Dyer [8], with hexane as single solvent for 8 h. Supercritical carbon dioxide (SCCO2) extraction
Bioprocess Biosyst Eng (2017) 40:911–918
Liquid CO2 from the storage tank (ST) is pressurized by a O2-pump and heated to the extraction temperature by a C heat exchanger (HEX 1). Subsequently, the CO2 flows in the extraction vessel (EX1) containing the biomass. The CO2/extract mixture is separated by controlled pressure reduction under simultaneous warming by heat exchangers (HEX2, HEX3) into a C O2 vapour and an extract phase (separators SP1, SP2). The extracts are removed from the process while the gaseous C O2 remains in the process. Afterwards, the CO2 is liquefied by a condenser and recirculated. All extractions were performed with sample sizes from 650 to 1300 g of lyophilized algae biomass. In terms of the high amount of biomass required for the extraction in the pilot plant, not all experiments could be carried out in duplicates. The online bentonite bleaching was only tested on the biomass of the unialgal S. obtusiusculus culture. The tests were focused on this strain, because it showed the most promising process parameters in terms of cultivation stability and biomass yields and therefore became the leading strain in the Advanced Biomass Value (ABV) project.
913 Table 1 Adapted temperature and pressure profiles of astaxanthin extractions, applied to mixed, lyophilized Scenedesmus biomass and the corresponding extraction yields after S CCO2 extraction and soxhlet extraction of the spent material Extraction pressure (MPa)
Extraction CO2: temp. (°C) biomass ratio
Extraction yield (% w/w)
Soxhlet yields of spent material (% w/w)
30 50 60 80
50 60 60 80
6.9 6.7 5.8 7.6
1.2 0.5 0.9 0.2
100 100 200 100
Extraction time 540 min
110 °C for 1 h, the oil was analyzed via inductively coupled plasma optical emission spectroscopy (ICP-OES) with a Perkin Elmer Optima 3300 DV, analog to DIN EN ISO 11885. The samples were dissolved in kerosene.
Results
Purification and analysis of microalgae extracts
Effect of pressure and temperature on extraction yield
Lipid analysis
The major points of regulation during the process of SCCO2 extraction are changes in extraction temperature and the applied pressure. For the first experiments, temperature and pressure profiles established in the pilot plant from astaxanthin extraction were tested on the lyophilized biomass of the mixed Scenedesmus culture (Table 1). The different temperature and pressure profiles resulted in comparable quantities of extracted lipids, but showed large differences in the quality of the extracts. The extracts obtained from the profiles with lower temperature and pressure were unclear, greenish-brownish, viscous liquids, whereas the extracts gained from the profiles with high temperature and pressure appeared as very dark brownish, almost black and extremely viscous substance. For most of the downstream applications of the extracted algae lipid fractions, a clear and homogeneous extract is required. In terms of developing an economically and ecologically balanced extraction process and to obtain a more clear and homogeneous extract, the following experiments were carried out under milder conditions (Table 2). The milder extraction conditions applied to the biomass samples resulted in lipid yields between 6.5 and 8.3% (w/w), suggesting that there is no statistically relevant distinction in the extraction efficiency compared to the extraction profiles applied before (Table 1). Although equal quantities were obtained, the quality of the extracts strongly improved. The obtained extracts are clear, homogenous liquids with slightly different colors. The extracts from the profiles 1 and 3 had a greenish color, while the extracts
The direct transesterification of the algae derived lipids was performed according to a modified protocol of Griffiths et al. [12] with the following modifications: replacement of the C17-TAG by a C12-TAG, replacement of BF3 methanol by a HCL-methanol solution, and the C19-ME was omitted. Subsequently, the resulting fatty acid methyl ester (FAME) extract was injected into a Thermo Scientific™ TRACE™ Ultra Gas Chromatograph coupled to a Thermo DSQ™ II mass spectrometer and the Triplus™ Autosampler injector. Column: Stabilwax® fused silica capillary (30 m × 0,25 mm, film thickness 0.25 μm). (Program: initial column temperature 50 °C, increasing (4 °C/min) up to a final temperature of 250 °C. Carrier gas: hydrogen, flow rate 3.5 mL/min.) Peaks were identified by comparison to a marine oil standard (Restek) or by specific molecular masses detected. Purification of crude micro algae extracts The lipids were purified with an adsorbent based on montmorillonite. In a column with n-hexane 20 g Tonsil 5 10® from Clariant Produkte (Deutschland) GmbH were filled and given 10 min to swell in the solvent. 2–3 g algae oil were applied to the column and given time to sink completely into the adsorbent. The elution was done with n-hexane until 150 ml eluate was collected. After removing the solvent with a rotary evaporator and drying the oil at
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Table 2 Different extraction profiles applied to mixed, lyophilized Scenedesmus biomass and the corresponding extraction yields after S CCO2 extraction and soxhlet extraction of the spent material
Profile
Extraction pressure (MPa)
Extraction temp. (°C)
CO2: biomass ratio
Extraction yield (% w/w)
Soxhlet yields of spent material (% w/w)
1 2 3 4
7 7 12 12 12 15 15
20 20 20 20 20 20 20
20 100 20 100 100 100 100
6.5 6.6 6.6 8.3 7 6.6 6.5
1.0 2.0 1.8 2.1 2.8 3.0 2.8
5
Extraction time 540 min
Table 3 Extraction parameters applied to Scenedesmus obtusiusculus biomass (unialgal culture) and the corresponding extraction yields after SCCO2 extraction and soxhlet extraction of the spent material (in duplicate) Extraction pressure (MPa)
Extraction CO2: temp. (°C) biomass ratio
Extraction yield (% w/w)
Soxhlet yields of spent material (% w/w)
12 12
20 20
6.4 6.4
0.5 0.9
100 100
Extraction time 540 min
As a proof of concept the extraction profile was applied to an unialgal S. obtusiusculus culture (Table 3) and was used to monitor the extraction efficiency (Fig. 1). The transferability of the extraction profile to other microalgae species within the Scenedesmus family is depicted in Table 3. Both quantity and quality of the extracts were comparable to those of the mixed S. obliquus culture extracted previously. The extraction curves in Fig. 1 depict the extraction yields (in % w/w) of four different samples, two samples of a mixed S. obliquus culture (square markers) and two samples of an unialgal S. obtusiusculus culture (triangle markers), as a function of the C O2 to biomass ratio. The four extraction curves follow a sigmoidal function and are almost reaching a plateau of maximum lipid yield at a ratio of 100 (CO2/biomass), matching the results shown in Table 1. In addition, the technical duplicates of the extraction curves demonstrate that the extraction method is reproducible. Microalgae extract analysis Fatty acid composition of extracts obtained from different extraction profiles
Fig. 1 Algae lipid extraction yields from two samples of a mixed Scenedesmus obliquus culture (square markers) and from two samples of an unialgal Scenedesmus obtusiusculus culture (triangle markers), expressed as a function of increasing C O2 to biomass ratio. Extraction time 840 min
from the other three profiles showed a more brownish to orange color. The extraction profile 4 showed the best results in this experimental set-up, with respect to the extraction effency and sustainability, and was employed for subsequent extractions.
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The analysis of the fatty acid (FA) composition of the microalgae extracts (Table 4) showed no significant difference for the extraction profiles tested. The major compounds in these extracts are linolenic acid (C18:3), hexadecatetraenoic acid (C16:4), linoleic acid (C18:2), oleic acid (C18:1) and palmitic acid (C16:0). The FA profile of the unialgal culture of S. obtusiusculus was similar to the one from the mixed S. obliquus culture in its major components, but the percentage of FAs identified from the total lipid extract is reduced by 20–30%. The obtained FA profiles accord with previously published profiles.
Bioprocess Biosyst Eng (2017) 40:911–918
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Table 4 Major fatty acid (FA) components in microalgae extracts from different extraction profiles, expressed as percentage of the total lipid extract (mean ± standard deviation) S. obliquus
S. obtusiusculus
Profile Pressure (MPa) CO2: biomass ratio C14 C16 C16:1 cis ω7 C16:1 ω9 C16:2 ω6 C16:3 ω3 C16:4 ω3 C18 C18:1 ω9 C18:1 ω7 C18:2 ω6 C18:3 ω3 C18:4 ω3
1 7 20 0.29 ± 0.01 6.30 ± 0.19 0.72 ± 0.02 1.40 ± 0.06 1.24 ± 0.04 2.31 ± 0.09 9.81 ± 0.33 0.13 ± 0.00 6.59 ± 0.14 1.20 ± 0.02 9.04 ± 0.18 24.78 ± 0.44 2.26 ± 0.09
2 7 100 0.27 ± 0.02 6.38 ± 0.09 0.72 ± 0.01 1.83 ± 0.02 1.31 ± 0.02 2.54 ± 0.02 11.93 ± 0.02 0.15 ± 0.00 7.02 ± 0.08 1.28 ± 0.01 9.75 ± 0.11 27.44 ± 0.24 2.81 ± 0.02
3 12 20 0.27 ± 0.01 6.46 ± 0.22 0.74 ± 0.03 1.53 ± 0.05 1.27 ± 0.04 2.28 ± 0.04 9.56 ± 0.55 0.14 ± 0.00 6.93 ± 0.23 1.25 ± 0.04 9.18 ± 0.05 24.75 ± 0.59 2.25 ± 0.12
4 12 100 0.29 ± 0.01 6.01 ± 0.05 0.74 ± 0.03 2.16 ± 0.03 1.43 ± 0.02 2.77 ± 0.03 13.25 ± 0.09 0.16 ± 0.00 7.43 ± 0.08 1.38 ± 0.04 10.20 ± 0.07 28.12 ± 0.16 3.02 ± 0.03
12 100 0.30 ± 0.01 5.68 ± 0.03 0.78 ± 0.02 2.03 ± 0.02 1.48 ± 0.07 2.77 ± 0.03 13.28 ± 0.44 0.15 ± 0.01 7.45 ± 0.07 1.38 ± 0.01 10.21 ± 0.08 28.44 ± 0.28 3.07 ± 0.01
5 15 100 0.29 ± 0.02 6.56 ± 0.09 0.77 ± 0.01 2.01 ± 0.00 1.44 ± 0.02 2.72 ± 0.02 12.66 ± 0.23 0.15 ± 0.00 7.24 ± 0.09 1.34 ± 0.02 9.94 ± 0.15 27.69 ± 0.35 2.96 ± 0.05
15 100 0.29 ± 0.02 6.07 ± 0.02 0.77 ± 0.01 1.89 ± 0.01 1.41 ± 0.01 2.70 ± 0.01 12.53 ± 0.16 0.14 ± 0.00 7.25 ± 0.07 1.34 ± 0.03 9.94 ± 0.10 27.75 ± 0.07 2.95 ± 0.01
C20 C20:1 ω9 FA yield
ND 0.05 ± 0.04 65.72 ± 1.58
ND 0.10 ± 0.01 73.53 ± 0.37
ND 0.05 ± 0.04 66.66 ± 0.83
0.03 ± 0.04 0.08 ± 0.00 77.07 ± 0.48
0.02 ± 0.00 0.08 ± 0.01 77.09 ± 0.87
0.02 ± 0.00 0.08 ± 0.01 75.87 ± 0.59
ND 0.10 ± 0.01 75.11 ± 0.09
4 12 100 0.16 ± 0.00 3.25 ± 0.05 0.45 ± 0.01 0.41 ± 0.03 0.71 ± 0.01 1.13 ± 0.02 3.91 ± 0.07 0.17 ± 0.01 4.09 ± 0.03 0.79 ± 0.00 6.93 ± 0.07 20.62 ± 0.29 0.88v ± 0.02 ND ND 43.52 ± 0.52
All extractions were carried out at 20 °C FA yield percentage of FAs from total lipids, ND not detectable
Table 5 Microalgae oils obtained from different extraction profiles before (crude) and after (purified) the processing with a bentonite
Processing of microalgae oils with bentonites The microalgae lipid fractions obtained from the mild extraction profiles show a higher quality then the ones obtained under harsh extraction conditions. Anyway the coloring of the extracts implements a relatively high amount of substances like chlorophylls and carotenoids. As
these substances could be problematic for any downstream usage of the microalgae oils, e.g., in technical applications, the lipid fractions were processed with the bentonite Tonsil 510®. The results of a single treatment of microalgae oils with the bentonite are shown in Table 5. After the processing the lipid fractions appear as a clear, liquid oil, suggesting that
13
Cleaned
